June E. Ayling, Ph.D.
University of California, Berkeley
Several of the most important cofactors in almost all organisms are based on elaborations of the pteridine molecule, which consists of two fused six-membered rings containing 4 nitrogen atoms. In mammals these fall into three classes: the tetrahydrofolates, tetrahydrobipoterin, and molybdopterin. All serve many critical roles in human metabolism, and while folate is an essential nutrient, e.g. a vitamin, the latter two can be synthesized within the body. Our laboratory has focused on the chemistry, biochemistry and clinical applications of the folates and tetrahydrobiopterin.
Poor folate status has been implicated in many disorders such as anemia and birth defects, but many aspects of its role in health and disease are still not understood. Folates are compounds in which the pteridine ring is attached to a p-amino-benzoylglutamate group, and much of the work in our laboratory is aimed at discovering new facets of this cofactor family. Folate coenzymes participate in the biosynthesis of three of the four DNA bases. Folate, together with vitamin B12, also maintains S-adenosylmethionine, the central methylation reagent in the body. A key to understanding the physiological effects of folate is recognizing that folic acid (a.k.a. pteroylglutamic acid) is not found to a significant extent in fresh tissues or foods, and as such has no known direct biological function. The active folate cofactors are all tetrahydrofolate and its so-called one-carbon derivatives. While folic acid can be converted to tetrahydrofolate by two successive iterations of the enzyme dihydrofolate reductase, we have demonstrated that this is very slow in humans in comparison to other animals. As a result, ingestion of folic acid even in the amount of the Recommended Daily Intake (0.4 mg) results in transient though appreciable appearance of this form still in its original unmetabolized state. Moreover, a small portion of this still unreduced folic acid remains long after the clearance of the bulk of the dose. This fasting unmetabolized folic acid is present in virtually all subjects examined in the United States. We propose that this is due to the constant exposure of those living in countries having mandatory fortification of the basic grain supply with folic acid, such as the U.S. Our laboratory is investigating whether the constant background of unmetabolized folic acid may have unanticipated consequences.
Pharmacologic doses of folic acid (1 to 5 mg/d) have been commonly used in intervention trials testing whether B-vitamins can also decrease the risk of cardiovascular disease or Alzheimer's diseases, in addition to decreasing neural tube affected births. We have found that as the dose is increased above 1 mg, dihydrofolate reductase becomes increasingly saturated, and the preponderance of folate in the plasma remains as unactivated folic acid for many hours. We are investigating means of administering folate that overcome this bottleneck. The mechanism of the differential effects of the highly bioavailable active forms of folate is a major focus of our current efforts.
For decades the role of folate in mammals has been thought to be primarily to aid in the transfer of one-carbon units (at various oxidation levels) from one molecule to another. Our laboratory has recently discovered a new property of 5-methyltetrahydofolate (5-MTHF), one of the most abundant forms in blood and tissues. We observed that 5-MTHF quenches the excited state of light activated photosensitizers at a nearly diffusion limited rate. It also rapidly reacts with any singlet oxygen that might be produced by ultraviolet light driven photosensitization reactions. In so doing, this form of folate can help to protect DNA from cleavage by UVA or UVB. This has considerable implications for the biology of folate in the skin, which we are currently investigating.
Curiously, 5-MTHF only slowly reacts with superoxide. Therefore, given this selectivity, it was uncertain how well 5-MTHF and other tetrahydrofolates might intercept hydroxyl radicals, one of the main intermediaries of damage by ionizing radiation. Surprisingly, both 5-MTHF and almost to the same extent the 5-formyl analog, were found to be excellent scavengers of hyrodxyl radicals. Since many cell types have the capacity to rapidly take up folate via the reduced folate carrier, experiments are being conducted to examine the protective effects of these natural forms of folate during exposure to x-rays and other types of ionizing radiation. Further, increased folate status may also aid in promoting DNA repair subsequent to irradiation, due to enhanced production of nucleotide bases. We are further exploring the application of these properties to help prevent and mitigate the molecular damage that can result during x-ray fluoroscopy and other exposures to radiation.
Thus, many of our goals are largely oriented to understanding the physiological impacts of the natural folates in comparison to synthetic folic acid and whether there are biological functions of these versatile tetrahydrofolate cofactors yet to be elucidated.
Tetrahydrobiopterin (BH4), serves as a cofactor for the three aromatic amino acid hydroxylases. Tryptophan and tyrosine hydroxylases are the initial and rate limiting steps in the biosynthesis of serotonin and the catecholamine neurotransmitters, respectively. Phenylalanine hydroxylase, which is deficient in the genetic disorder PKU, provides the only efficient means of metabolizing excess dietary intake of phenylalanine. For these hydroxylases, BH4 participates in the utilization of dioxygen. The O2 molecule is somewhat unusual in that it is in the triplet state at room temperature. This fortunate quantum mechanical oddity prevents organic matter (e.g., humans) from spontaneous combustion. On the other hand, life has been required to evolve mechanisms (of which there are several) to activate O2 as part of oxidative metabolism. We have studied the role of BH4 in this activation process which also involves an atom of non-heme iron that is found in all three hydroxylases. This function of BH4 is distinct from its mechanism in the nitric oxide synthase reaction where it participates chemically as a one-elecron donor to the heme domain. The natural isomer of 6R-BH4 is biosynthesized starting from GTP. Early investigations of hydroxylase mechanism were facilitated by our isolation and characterization of the natural and unnatural diastereoisomers of BH4. Our interest in the stereospecific cofactor properties of BH4 eventually led us to develop the first, and still only, total stereospecific synthesis of tetrahydropteridines, which we then examined as hydroxylase cofactors. This versatile method incorporates intact the chirality of an amino acid precursor into the structure of the final tetrahydropteridine. The usual catalytic reduction of fully oxidized pteridines typically yields tetrahydro-products that are racemic at the 6-position.
This synthetic methodology, which involves an oxidative cyclization via a pterin-4a-hydroxy intermediate, was put to another use - to study the mechanism of one of the enzymes that helps to regenerate BH4 after its oxidation during any of the hydroxylase reactions. The function of this enzyme, 4a-hydroxy-tetrahydropterin dehydratase (PCD), is nominally only the removal of a molecule of water from the oxidized BH4 hydroxylase reaction. Despite this very simple reaction, PCD has an additional function: that of the transcription cofactor DCoH that interacts with and stabilizes the transcription factor, HNF1. An outstanding question in this area is whether the bifunctionality of this protein is purely adventitious, or might be related to some as yet unidentified nexus between BH4 pathways and HNF1 activity.
Recent evidence has been pointing toward some overlap between BH4 and 5-MTHF in regulating vascular endothelial function. Understanding the properties of both of these pteridine cofactors will facilitate deconvolving this relationship.
- Activity of the Bifunctional Protein 4a-Hydroxy-tetrahydropterin Dehydratase/DCoH during Human Fetal Development: Correlation with Dihydropteridine Reductase Activity and Tetrahydrobiopterin Levels. I. Rebrin, S.W. Bailey and J.E. Ayling. Biochem. Biophys. Res. Comm. 217, 958-965 (1995).
- Catalytic Characterization of 4a-Hydroxy-tetrahydropterin Dehydratase. I. Rebrin, S.W. Bailey, S.R. Boerth, M.D. Ardell and J.E. Ayling. Biochemistry 34, 5801-5810 (1995).
- Synthesis of 4a-Hydroxy-tetrahydropterins and the Mechanism of their Non-enzymatic Dehydration to Quinoid Dihydropterins. S.W. Bailey, I. Rebrin, S.R. Boerth and J.E. Ayling. J. Am. Chem. Soc. 117, 10203-10211 (1995).
- Mechanism of Oxygen Activation by Phenylalanine Hydroxylase. M. D. Ardell, S. W. Bailey, and J. E. Ayling. Chemistry and Biology of Pteridines and Folates (Proceedings of the 11th International Symposium). Eds. W. Pfleiderer and H. Rokos. Blackwell Science, Berlin pp. 491-496 (1997).
- Total Chemical Synthesis of Chirally Pure (6S)-Tetrahydrofolic Acid. S.W. Bailey and J.E. Ayling. Methods in Enzymology 281, 3-16 (1997)
- Mechanism of Dehydration by the Bifunctional Protein, 4a-Hydroxy-tetrahydropterin Dehydratase/DCoH. J.E. Ayling, I. Rebrin, B. Thöny, and S.W. Bailey. Chemistry and Biology of Pteridines and Folates. Eds. W. Pfleiderer and H. Rokos. Blackwell Science, Berlin, 565-570 (1997).
- Mechanistic Studies of 4a-Hydroxytetrahydropterin Dehydratase from Pseudomonas aeruginosa. I. Rebrin, J. Song, R.A. Jensen, and J.E. Ayling. Chemistry and Biology of Pteridines and Folates. Eds. W. Pfleiderer and H. Rokos. Blackwell Science, Berlin, 631-634 (1997).
- Molecular Modeling Lectures over the Internet. J.D. Madura, L. Laaksonen, and J.E. Ayling. American Chemical Society 216, COMP27 (1998).
- Stereospecificity and Catalytic Function of Histidine Residues in 4a-Hydroxy-tetrahydropterin Dehydratase/DCoH. I. Rebrin, B. Thöny, S.W. Bailey, and J.E. Ayling. Biochemistry 37, 11246-11254 (1998).
- Transient Hyperphenylalaninemia with High Levels of 7-Biopterin is Associated with Mutations in the PCBD Gene Encoding the Bifunctional Protein Pterin-4a-Carbinolamine Dehydratase and Transcriptional Coactivator (DCoH). B. Thöny, F. Neuheiser, L. Keirat, M. Blaskovics, P.H. Arn, P. Ferreira, I. Rebrin,J.E. Ayling, and N. Blau. American Journal of Human Genetics 62, 1302-1311 (1998).
- Mutations in the Pterin-4a-Carbinolamine Dehydratase Gene (PCBD) are Causative for a Benign Form of Hyperphenylalaninemia. B. Thöny, F. Neuheiser, L. Kierat, M.O. Rolland, P. Guibaud, T. Schlüter, R. Germann, R.A. Heidenreich, M. Duran, J.B.C. de Klerk, J.E. Ayling, and N. Blau. Human Genetics 103, 162-167 (1998).
- Hyperphenylalaninemia and 7-Pterin Excretion Associated with Mutations in 4a-Hydroxy-tetrahydrobiopterin Dehydratase/DCoH: Analysis of Enzyme Activity in Intestinal Biopsies. J.E. Ayling, S.W. Bailey, S.R. Boerth, R. Giugliani, C. Braegger, B. Thöny and N. Blau. Molecular Genetics and Metabolism 70, 179-188 (2000).
- Hyperphenylalaninemia Associated with Mutations in 4a-Hydroxy-tetrahydropterin Dehydratase/DCoH: Analysis in Intestinal Biopsies. J.E. Ayling, S.R. Boerth, S.W. Bailey, R. Giugliani, C. Braegger, B. Thony and N. Blau. Pteridines, (2000).
- Crystal Structure of 4a-OH-Tetrahydropterin Dehydratase/DCoH Complexed with a Substrate Analog. L.Q. Chen, P.H. Roberts, E.J. Meehan, K.L. Gross, S.W. Bailey, J.E. Ayling. American Crystallographic Association, W0133, (2000).
- An HPLC Method for Analysis of Unreduced Folic Acid in Blood. S.W. Bailey, M.R. Malinow and J.E. Ayling. FASEB J. 14, LB204, (2000).
- Hyperphenylalaninemia Due to Pterin-4a-carbinolamine Dehydratase: Analysis of Intestinal Biopsies. N. Blau, S.R. Boerth, S.W. Bailey, R. Giugliani, C.P. Braegger, B. Thony and J.E. Ayling. J. Inherited and Metabolic Diseases, 23, (2000).
- Stereospecific Synthesis of 2-Desamino-tetrahydropterins as Probes of Hydroxylase Cofactor Recognition. S. Vasudevan, S.W. Bailey, and J.E. Ayling. Pteridines 12, 91 (2001).
- Stereospecific Synthesis of 2-Desamino-tetrahydropterins as Probes of Hydroxylase Cofactor Recognition. S. Vasudevan, S.W. Bailey, and J.E. Ayling. Chemistry and Biology of Pteridines and Folates. Ed. S. Milstien. Kluwer Academic Publ. 37-41 (2002).An Assay for Dihydrofolate Reductase in Human Tissues by HPLC with fluorometric Detection. S.W. Bailey, M.C. Syslo, and J.E. Ayling. FASEB Journal 16, A267 (2002).
- The Fate of Oxidized 5-Methyltetrahydrofolic Acid. S.W. Bailey, E.A. Sabens, and J.E. Ayling. FASEB Journal 17, A310 (2003).
- Unreduced Folic Acid in Plasma of Subjects Consuming Either Folic Acid or 5-Methyl-tetrahydrofolate. S.W. Bailey, M.R. Malinow, D.L. Hess, P.B. Duell, B.M. Upson. E.G. Graf, A. Irvin-Jones, M.C. Syslo, and J.E. Ayling. FASEB Journal 17, A311 (2003).
- Folic Acid Pharmacokinetics: Dose Dependent Metabolism. S.W. Bailey, M.R. Malinow, D.L. Hess, B.M. Upson. E.G. Graf, M.V. Cohen, M. Nozawa, P.B. Alverson and J.E. Ayling. J. Inherited and Metabolic Diseases 26, (Suppl 1) 122 (2003).
- An Assay for 5-Methyl-tetrahydrofolate in Urine. S.W. Bailey, M. Nozawa, J.E. Ayling. FASEB Journal 18, A176 (2004).
- Dose Dependent Lack of Metabolism in Humans. S.W. Bailey, M.R. Malinow, D.L. Hess, B.M. Upson, E. Graf, M..V.Cohen, M. Nozawa, P.B. Alverson, J.E. Ayling. FASEB J. 18, A176 (2004).
- Urinary folate Excretion after Oral Doses of Folic acid or 5-Methyl-6(S)-tetrahydrofolate in Humans. S.W. Bailey, M. Nozawa, P.B Alverson, M.V. Cohen and J.E. Ayling. FASEB J. 19, A52 (2005).
- Pharmacokinetics of Oral Folic Acid Compared to 5-Methyl-6(S)-tetrahydrofolate in Human Plasma. S.W. Bailey, C.M.. Pfeiffer, M. Zhang, P. B. Alverson, M. Nozawa, M.V. Cohen, and J.E. Ayling. FASEB Journal 19, A52 (2005).
- Catalytic Efficiency of Dehydratase/DCoHalpha, an Isozyme of Human 4a-Hydroxy-tetrahydropterin Dehydratase/DCoH. S.W. Bailey, J.M. Hevel, and J.E. Ayling. Pteridines 16, 94 (2005).
- Interaction of Folate and Radiation. S.W. Bailey, T.Offer,, E.A. Sabens, J.E. Ayling, and B.N. Ames. Pteridines 16, 65 (2005).
- An Acute Decrease in Plasma Total Homocysteine Promoted Specifically by a Single Dose of 5-Methyl-6(S)-tetrahydrofolate. S.W. Bailey, R.M. Malinow, B.M. Upson, E.Graf, C.M. Pfeiffer, M.Zhang, M.Nozawa, P.B. Alverson, M.V. Cohen, D.T. Redden, and J.E. Ayling. Haem. Reports 1, 17 (2005).
- Can the DCoHalpha Isozyme Compensate in Patients with 4a-Hydroxy-tetrahydrobiopterin Dehydratase/DCoH Deficiency? J.M. Hevel, J.A. Stewart, K.L. Gross and J.E. Ayling. Molecular Genetics and Metabolism 88, 38-46 (2006).
- Acute and Chronic Effects of Folic Acid and [6S]-5-Methyltetrahydrofolate on Blood Pressure and Endothelial Function. S.W. Bailey, N. Mann, T. Tamura, M.V. Cohen, and J.E. Ayling. Clinical Chemistry and Laboratory Medicine 45, 26 (2007).
- Fasting Plasma Levels of Unmetabolized Folic acid in Human Subjects after Chronic Treatment with Pharmacologic Doses of Folates. S.W. Bailey, P.B. Alverson, M.. Nozawa, M.V. Cohen and J.E. Ayling. FASEB J. 21, 545.3 (2007).
- 5-Methyltetrahydrofolate inhibits photosensitization reactions and strand breaks in DNA. T. Offer T, B.N. Ames, S.W. Bailey, E.A. Sabens, M. Nozawa, and J.E. Ayling. FASEB J. 21, 2101-2107 (2007).
- Determinants of Oligomerization of the Bifunctional Protein DCoH_ and the Effect on its Enzymatic and Transcriptional Coactivator Activities. J.M. Hevel, P. Pande, S.Viera-Oveson, T.J. Sudweeks, L. S. Jaffree, C. M. Hansen, and J E. Ayling. Archives Biochem. Biophys. 477, 356-362 (2008).Protection of DNA from Radiation Damage by the Predominant Folate in the Circulation:5-Methyl-tetrahydrofolate. S.W. Bailey, K. Lenton and J.E. Ayling. IRPA 12 Proc., TS1.2.1-2397 (2008).
- The dose dependent effect of chronic folate administration on S-adenosylmethionine and S-adenosylhomocysteine in human plasma. S.W. Bailey, P.B. Alverson, M.V. Cohen and J.E. Ayling. FASEB J. 23, 557.2 (2009).
- Differential response to folic acid versus natural 5-methyltetrahydrofolate in humans. S.W. Bailey and J.E. Ayling. Pteridines (2009).
- Differential alteration of plasma thiols and S-adenosylmethionine upon treatment with high dose folic acid or 5-methyltetrahydrofolate. S.W. Bailey, H. Refsum, O.A. Baarholm, T. Tamura, M.V. Cohen, P.B. Alverson and J.E. Ayling. Proceedings of the 7th International Conference on Homocysteine Metabolism, 53 (2009).
- Prophylaxis of damage from ionizing and ultraviolet radiation by the natural folate 5-methyltetrahydro-folic acid. S.W. Bailey, K. Lenton and J.E. Ayling. Health Physics Society Congress TAM-F. 7 (2009).
- Persistent circulating unmetabolised folic acid in a setting of liberal voluntary folic acid fortification. Implications for further mandatory fortification? M.R. Sweeney, A. Staines, L. Daly, A. Trainor, S. Daly, S.W. Bailey, P.B. Alverson, J.E. Ayling, and J.M. Scott. BMC Public Health. 9, 295 (2009)
- The extremely slow and variable activity of dihydrofolate reductase in human liver and its implications for high folic acid intake. S.W. Bailey and J.E. Ayling. Proc. Nat. Acad. Sci. USA. 106 15424-9 (2009).
- A surprising level of unmetabolized folic acid in serum from the Tsimane Amerindians of Bolivia. S.W. Bailey, M. Gurven, H. Kaplan, P.B. Alverson and J.E. Ayling. FASEB J. 24: 228.8 (2010).
- Interconversions of plasma folate species in response to dosing with oral folic acid and 5-methyltetrahydrofolate: an LC-MS/MS pilot study. Z. Fazili, S.W. Bailey, N. Paladugula, J.E. Ayling and C.M. Pfeiffer. FASEB J. 24: 915.1 (2010).
- No correlation between unmetabolised folic acid levels and folate gene polymorphisms in an Irish population. MacCooey A, Sweeney MR, Boilson A, Scott JM, Staines A, Kelleher C, Daly LL, Bailey SW, Alverson PB, Ayling JE, Parle-McDermott A. Irish Society of Human Genetics, Belfast, Ireland. Ulster Med J. Sep:80(2010).
- A potential new role for the B vitamin 5-methyl-tetrahydrofolate in human skin. Hasoun LZ, Bailey SW, Outlaw KK, Ayling JE. FASEB J;25:608-2(2011).
- Unmetabolized folic acid prevalence is widespread in the older Irish population despite the lack of a mandatory fortification program. Boilson A, Staines A, Kelleher CC, Daly L, Shirley I, Shrivastava A, et al. Am J Clin Nutr;96(3):613-21(2012).
- Rapid folate repletion as an additional option for prevention of birth defects. Bailey SW, Ayling JE. Journal of Clinical Chemistry and Laboratory Medicine;50(2):A20-1(2012).
- Elevation of folate status in humans by 5-methyl-6S-tetrahydrofolate. Bailey SW, Ayling JE. FASEB J;26:1020(2012).
- Effect of serum folate status on total folate and 5-methyltetrahydrofolate in human skin. Hasoun LZ, Bailey SW, Outlaw KK, Ayling JE. Am J Clin Nutr;98(1):42-8(2013).
Folate in human skin: its correlation with serum levels, and the unusual abundance in the epidermis of 5-methyltetrahydroflate. Hasoun LZ, Bailey SW, Outlaw KK, Ayling JE. FASEB J;27:1077.2(2013).
See Dr. Ayling's PubMed listing